The long-term objective of this proposal is to determine the mechanism by which the packaging of a gene in heterochromatin leads to stable inactivation. This type of """"""""off"""""""" regulation is clearly of functional important in chromosome imprinting, and may extend to the regulation of the homeotic loci, whose on/off pattern is critical for normal develop- ment in all higher organisms. Drosophila will be used throughout this study because of the ease of manipulation and monitoring of Position effect variegation, one type of epigenetic down-regulation that appears to reflect the packaging of the test gene. We have identified a heterochromatin-associated protein, HP1, which plays a role in this gene inactivation; mutations in HP1 result in suppression of position effect variegation in Drosophila.
Our first aim i s to identify proteins which interact with HP1, using biochemical and genetic approaches. Interacting proteins will be sought using (a) a genetic screen in Drosophila; (b) coprecipitation with anti-HP1 antibodies; and (c) a screen in yeast that requires protein-protein interactions to score a positive result (Fields and Song, 1989, Nature 340, 245-246). In the genetic screen, missense mutations, including temperature-sensitive alleles of HP1, are being sought, as well as noncomplementing second-site mutations. Interacting proteins identified will be characterized biochemically and genetically to determine their role in heterochromatin formation. In parallel with the study of HP1, we are carrying out studies of the nucleosomal and higher order packaging of transgenes inserted into heterochromatin using P-element mediated transformation. Two different genes are being used, hsp26, a heat shock gene whose 5' upstream regulatory region is normally preset in an accessible chromatin structure prior to gene activation, and a CAT reporter gene under the control of glucocorticoid response elements, a gene likely to be characteristic of those whose 5' upstream regulatory region is packaged in a nucleosomal array and must be remodeled for activation. We will characterize the chromatin structure of these genes when they are packaged in heterochromatin, in comparison to their packaging in euchromatin, in order to learn more about the inactivation process. Having such specific test genes in a heterochromatin environment will also be invaluable in assessing the role of the chromosomal proteins identified above. These studies should move us much closer to an understanding of the nature and regulatory role of higher order chromatin structure.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD023844-06A1
Application #
3324167
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1987-12-01
Project End
1998-06-30
Budget Start
1993-07-15
Budget End
1994-06-30
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Arts and Sciences
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Pal-Bhadra, Manika; Leibovitch, Boris A; Gandhi, Sumit G et al. (2004) Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303:669-72
Shaffer, Christopher D; Stephens, Gena E; Thompson, Brandi A et al. (2002) Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin. Proc Natl Acad Sci U S A 99:14332-7
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Cryderman, D E; Cuaycong, M H; Elgin, S C et al. (1998) Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin. Chromosoma 107:277-85

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